WO2021170111A1 - Activateur immunitaire tumoral, son procédé de préparation et son utilisation - Google Patents

Activateur immunitaire tumoral, son procédé de préparation et son utilisation Download PDF

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WO2021170111A1
WO2021170111A1 PCT/CN2021/078264 CN2021078264W WO2021170111A1 WO 2021170111 A1 WO2021170111 A1 WO 2021170111A1 CN 2021078264 W CN2021078264 W CN 2021078264W WO 2021170111 A1 WO2021170111 A1 WO 2021170111A1
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tumor
amino acid
polypeptide
immune enhancer
tumor immune
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PCT/CN2021/078264
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English (en)
Chinese (zh)
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邓立
陈淑娴
曲秀霞
龚笑海
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无锡派列博生物医药科技有限公司
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Priority to CA3169907A priority Critical patent/CA3169907A1/fr
Priority to US17/907,941 priority patent/US20230045104A1/en
Priority to AU2021226544A priority patent/AU2021226544B2/en
Priority to EP21761114.4A priority patent/EP4112636A4/fr
Priority to JP2022552322A priority patent/JP7513303B2/ja
Publication of WO2021170111A1 publication Critical patent/WO2021170111A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00119—Melanoma antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants

Definitions

  • the present invention relates to the field of immunity, and more specifically to a tumor immune enhancer and its preparation method and application.
  • the root cause of tumor development is that the genetic material of tissue cells has mutations that promote abnormal cell growth. Although the number of gene mutations in tumor cells can be as many as thousands, the mutated genes (driver genes) that actually initiate tumorigenesis may only involve two to three. Part of the driver genes have been used as targets for tumor therapy drug design. Studies have found that the clinical efficacy of such targeted drugs depends on the expression of other non-driver mutant genes and untargeted driver genes in tumor cells, so the clinical efficacy of such drugs is very limited. The latest research progress of tumor vaccines shows that we can design vaccines that can cure tumors by using gene mutations that change the encoding of amino acid residues. This is an important milestone in tumor treatment.
  • the gene encoding the mutation may be broken down by the antigen-presenting cells to produce a tumor-specific antigen that can be recognized by T cells.
  • This antigen is called a tumor neoantigen. Since T cells that recognize neoantigens are not restricted by central tolerance, neoantigens are considered to have strong immunogenicity and can stimulate tumor-specific killing CD8+ T cell (CTL) responses. Clinical studies have confirmed that such neoantigen-specific CTLs do exist in tumor tissues, and the activity of such CTLs determines a variety of tumor treatment methods such as T cell growth factor IL-2, immune checkpoint inhibitors (PD-1 and PD-1). CTLA-4 monoclonal antibody) and the efficacy of infusion of expanded TILs in vitro.
  • neoantigens of most tumors are patient-specific, vaccines against neoantigens must be customized according to the genetic mutations of each patient.
  • genetic mutations in a single tumor have polyclonal properties.
  • tumor vaccines need to adopt a multi-target design strategy.
  • the number of neoantigen T epitopes used in the published clinical studies of tumor vaccines is more than 20.
  • the detection of CD4 and CD8 T cell responses revealed that only about one-third of the neoantigens can trigger anti-tumor T cell responses, and more than 90% of T cells are CD4 + .
  • Type I helper T cells are required for such cell activation and memory formation, as well as tumor infiltration. Therefore, it can be inferred that most of the neoantigens currently used in cancer vaccines cannot effectively trigger type I helper T cell responses.
  • T helper polypeptides For example, tetanus toxoid T helper peptide and pan-DR peptide (PARDE) have been used in clinical trials. As a result, it was found that these polypeptides could not effectively induce anti-tumor CD8+CTL responses.
  • PARDE pan-DR peptide
  • the purpose of the present invention is to provide an effective anti-tumor immune response immune enhancer and its preparation method and application.
  • a tumor immune enhancer which comprises one or more polypeptides having the structure of Formula I, or a pharmaceutically acceptable salt thereof:
  • Z0 is none, or a peptide consisting of 1-10 amino acid residues
  • Z2 is none, or a peptide consisting of 1-10 amino acid residues
  • Z1 is a peptide selected from the following group:
  • amino acid sequence of SEQ ID NO: 1-9 is formed by substitution, deletion or addition of one, two or three amino acid residues, and is capable of binding to human and mouse type II histocompatibility antigens. .
  • the derivative polypeptide retains ⁇ 70% of the activity of the polypeptide shown in SEQ ID NO: 1-9 to bind to human and mouse type II histocompatibility antigens.
  • the identity of the derivative polypeptide with the polypeptide shown in SEQ ID NO: 1-9 is ⁇ 80%, preferably ⁇ 90%, and more preferably ⁇ 95%.
  • the IC 50 of the binding of Z1 to the histocompatibility antigen is 5-50 nm.
  • Z0 and Z2 are not null at the same time
  • Z0 and Z2 contain at least 2-8, preferably 3-6, hydrophilic amino acid residues.
  • Z2 is none
  • Z0 is a peptide consisting of 2-8, preferably 3-6 hydrophilic amino acid residues.
  • Z0 is none
  • Z2 is a peptide consisting of 2-8, preferably 3-6, hydrophilic amino acid residues.
  • the hydrophilic amino acid is selected from the group consisting of arginine and lysine.
  • At least one of Z0 and Z2 contains (Arg)n structure, where n is a positive integer of 3-6.
  • the structure of Formula I is a structure from N-terminus to C-terminus.
  • the structure of Formula I is a structure from C-terminus to N-terminus.
  • "a” is a covalent bond (such as a peptide bond), or when Z0 is absent, then the "-" between Z0 and Z1 does not exist, or when Z2 is absent, then Z1 and The "-" between Z2 does not exist.
  • the tumor immunity enhancer has the activity of enhancing tumor immunity.
  • the tumor immune enhancer can induce a killer T cell response.
  • polypeptide having the structure of Formula I contains one or more CD4 + Th epitope peptides.
  • polypeptide having the structure of Formula I is a CD4+ T cell antigen receptor ligand.
  • the tumor immune enhancer comprises Z1 being the polypeptide of formula I shown in SEQ ID NO: 1, Z1 being the polypeptide of formula I shown in SEQ ID NO: 2, and Z1 being SEQ ID NO: A polypeptide of formula I shown in 3.
  • a multimer which is formed by m monomers in series and has the function of enhancing tumor immunity, wherein m is a positive integer ⁇ 2, and the Each monomer independently has the structure of Formula I:
  • Z0 is none, or a peptide consisting of 1-10 amino acid residues
  • Z2 is none, or a peptide consisting of 1-10 amino acid residues
  • Z1 is a peptide selected from the following group:
  • n 2, 3, 4, 5, or 6.
  • the two monomers are directly connected by a peptide bond or connected by a peptide linker.
  • the peptide linker is a flexible peptide linker, a rigid peptide linker, or a combination thereof.
  • the peptide linker is a peptide linker of 3-10 amino acids.
  • an isolated nucleic acid molecule which encodes a polypeptide, the polypeptide has the structure of Formula I, or the polypeptide is a multimer formed by m monomers in series, wherein each The monomers each independently have the structure of formula I, and m is a positive integer ⁇ 2.
  • the nucleic acid molecule is selected from the group consisting of DNA, RNA or a combination thereof.
  • a pharmaceutical composition which contains:
  • the dosage form of the composition is an injection.
  • the composition is a sustained-release dosage form.
  • the tumor immune enhancer according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, or the multimer according to the second aspect of the present invention contained in the composition can be selected from From the form of the next group:
  • Protein molecules (complete molecules, subunits, domains, polypeptides and recombinant engineering molecules), lipids, polysaccharides, lipids and polysaccharide complexes.
  • the pharmaceutical composition further contains:
  • the tumor antigen is a tumor neoantigen peptide.
  • the tumor antigen is an antigen that cannot effectively trigger an anti-tumor immune response.
  • the tumor antigen is natural, artificially synthesized, or a combination thereof.
  • the tumor antigen is selected from the group consisting of short peptides, intact proteins, tumor cell lysates, inactivated tumor cells, or a combination thereof.
  • the tumor antigens are derived from malignant tumors with non-synonymous mutations in the middle and high frequency encoding genes, including but not limited to:
  • Malignant melanoma breast cancer, lung cancer, pancreatic cancer, prostate cancer, bowel cancer, liver cancer, esophageal cancer, cervical cancer, bladder cancer, renal cell carcinoma, glioma multiforme.
  • the pharmaceutical composition further contains:
  • the DC activator is a nano-formulation, including but not limited to w/o agent and o/w agent.
  • the DC activator is a self-assembled nano-formulation.
  • the DC activator is selected from the group consisting of liposomes, viral outer capsids, and inactivated bacteria.
  • the pharmaceutical composition is a tumor vaccine composition.
  • the vaccine is selected from the following group: whole cell vaccine, cell lysate vaccine, tumor tissue split vaccine, tumor cell exosomal vaccine.
  • the polymer, or the isolated nucleic acid molecule of the third aspect of the present invention, and the substance is used to prepare drugs that improve the anti-tumor activity of tumor antigens, or to prepare anti-tumor vaccines (tumor vaccines) )combination.
  • the drug or vaccine composition is also used for one or more purposes selected from the following group:
  • helper T cell response preferably a type I helper T cell response
  • a medicine kit containing:
  • the drugs for treating tumors include immune checkpoint inhibitors, chemotherapeutic agents, radiotherapy agents, hyperthermia agents, oncolytic viruses, and immune cell therapeutic agents.
  • the immune cell therapeutic agent includes CAR-T cells, CAR-NK cells, TCR-T cells, DC cells or a combination thereof.
  • a method for treating cancer including the steps:
  • steps (i) and (ii) can be performed simultaneously or sequentially.
  • the object in need refers to a tumor patient.
  • step (ii) the drug for treating tumors is administered according to the usual dosage and frequency of administration.
  • the method is to combine existing tumor treatment methods such as immune checkpoint inhibitors, chemotherapy, radiotherapy, hyperthermia, and oncolytic viruses with the tumor immune enhancer of the present application.
  • existing tumor treatment methods such as immune checkpoint inhibitors, chemotherapy, radiotherapy, hyperthermia, and oncolytic viruses.
  • the method is to combine cell therapy with the tumor immune enhancer of the present application.
  • cell therapies include but are not limited to DC cell therapy, CAR-T cell therapy, CAR-NK cell therapy, etc. Cell therapy.
  • a method for preventing and/or treating tumors in mammals comprising the steps of: administering the tumor immune enhancer according to the first aspect of the present invention or its pharmaceutically acceptable Salt, the multimer according to the second aspect of the invention, the isolated nucleic acid molecule according to the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention.
  • the subject is a human.
  • the tumor antigens are derived from malignant tumors with non-synonymous mutations in the encoding subtypes of medium and high frequencies, such as: melanoma, pancreatic cancer, prostate cancer, breast cancer, lung cancer, colon cancer, liver cancer, esophageal cancer, Cervical cancer, bladder cancer, renal cell carcinoma, glioma multiforme.
  • the method further comprises: administering a tumor antigen to a subject in need.
  • the method further comprises: administering a drug for treating tumors to a subject in need.
  • Figure 1 shows that in one embodiment of the present invention, UThE enhances the anti-tumor immune response of the weak neoantigen of mouse melanoma.
  • Figure 2 shows that in another embodiment of the present invention, UThE enhances the anti-tumor immune response (tumor prevention) of the weak neoantigen of mouse melanoma.
  • Figure 2A and Figure 2B respectively show the change in tumor volume with the number of days after tumor injection.
  • FIG. 3 shows that in another embodiment of the present invention, UThE effectively enhances the therapeutic effect on mouse melanoma.
  • Figure 4 shows the results of detection of anti-UThE antibodies in mouse serum samples after vaccine immunization in an example.
  • Figure 5 shows the results of anti-UThE antibody detection in mouse serum samples after vaccine immunization in another embodiment.
  • Figure 6 shows the detection results of anti-neoantigen antibodies in mouse serum samples after vaccine immunization in another embodiment.
  • Fig. 7 shows the detection results of anti-neoantigen antibodies in mouse serum samples after vaccine immunization in another embodiment.
  • Figure 8 shows the results of specific CD8+CTL detection of mouse spleen tumors after vaccine immunization in another example.
  • Figure 9 shows the results of tumor-specific CD8+CTL detection in the spleen of immunized mice in another example.
  • Figure 10 shows the effective killing effect of the killing CD8+CTL on tumor target cells in another example.
  • FIG 11 shows that in another example, UThE effectively enhances the immunogenicity of mouse lung cancer (LLC) weak antigens and suppresses tumors.
  • LLC mouse lung cancer
  • FIG 12 shows another example, UThE effectively enhances the immunogenicity of the weak antigen of mouse melanoma (B16F10) and its inhibitory effect on tumors.
  • FIG 13 shows another example, UThE effectively enhances the immunogenicity of the weak antigen of mouse prostate cancer (RM-1) and its inhibitory effect on tumors.
  • FIG 14 shows another example, UThE effectively enhances the immunogenicity of mouse pancreatic cancer (PanC-02) weak antigen and its inhibitory effect on tumors.
  • Figure 15 shows the dose-efficacy relationship and safety of UThE in killing tumor cells in another example (mouse weight is stable).
  • Figure 16 shows the tumor suppressor effect of UThE candidate peptide, UThE1-7, in C57BL/6 mice and Balb/C mice in another example.
  • the inventors discovered for the first time a general Th epitope peptide derived from diphtheria toxin and tetanus toxin protein, which can enhance the tumor neoantigen vaccine-specific CD8 + CTL immune response.
  • the polypeptides of the present invention can enhance the anti-tumor immune response efficacy of tumor vaccines. On this basis, the present invention has been completed.
  • UThE1-UThE9 are polypeptide molecules composed of 15-21 amino acid residues, have a moderate affinity with mouse type II histocompatibility antigen molecules, and have an IC50 of 5-50 nm. Because these polypeptide molecules are too hydrophobic, it is necessary to add multiple hydrophilic amino acid residues (such as arginine residues or lysine residues) at the N-terminus or C-terminus during synthesis. These polypeptide molecules all carry one or more Th epitopes, which can support the function of inducing helper T cell responses in more than 80% of the population.
  • UThE1-UThE9 are polypeptide molecules composed of 15-21 amino acid residues, have a moderate affinity with mouse type II histocompatibility antigen molecules, and have an IC50 of 5-50 nm. Because these polypeptide molecules are too hydrophobic, it is necessary to add multiple hydrophilic amino acid residues (such as arginine residues or lysine residues) at the N-terminus
  • polypeptide of the present invention refers to a polypeptide having the structure of Formula I:
  • Z0 is none, or a peptide consisting of 1-10 amino acid residues
  • Z2 is none, or a peptide consisting of 1-10 amino acid residues
  • Z1 is a peptide selected from the following group:
  • a derivative polypeptide that is formed by substituting, deleting or adding one, two or three amino acid residues to the amino acid sequence of SEQ ID NO: 1-9 and can bind to type II histocompatibility antigen.
  • the polypeptide of the present invention includes a variant form of SEQ ID NO: 1-9 that has the function of enhancing tumor immunity.
  • variant forms include (but are not limited to): 1-4 (preferably 1-3, more preferably 1-2, most preferably 1) amino acid deletion, insertion and/or substitution, and One or several (usually within 4, preferably within 3, more preferably within 2) amino acids are added or deleted at the C-terminal and/or N-terminal.
  • amino acids with similar or similar properties are substituted
  • the function of the protein is usually not changed.
  • adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein.
  • the term also includes the polypeptide of the invention in monomeric and multimeric forms.
  • the term also includes linear and non-linear polypeptides (such as cyclic peptides).
  • polypeptide molecule of the present invention is too hydrophobic, it is necessary to add multiple hydrophilic amino acid residues (such as arginine residues or lysine residues) at the N-terminus or C-terminus during synthesis.
  • a typical polypeptide of the present invention is to add 3-6 arginines to the N-terminus or C-terminus of the polypeptide shown in SEQ ID NO: 1-9.
  • the present invention also includes active fragments, derivatives and analogs of UTHE polypeptides.
  • fragment refers to polypeptides that substantially maintain the function or activity of enhancing tumor immunity.
  • polypeptide fragments, derivatives or analogs of the present invention can be (i) a polypeptide with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusing a DTHE polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a protein fused to a leader sequence, secretory sequence, or 6His tag sequence). According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • a preferred type of active derivative means that compared with the amino acid sequence of formula I, there are at most 4, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid is composed of amino acids with similar or similar properties. Replaced to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Preferred substitution Ala(
  • the present invention also provides analogs of DTHE polypeptides.
  • the difference between these analogs and the natural DTHE polypeptide may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.
  • the polypeptide of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include (but are not limited to) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include: salts with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), and in the form of esters, carbamates or other conventional "prodrugs".
  • the present invention provides a tumor immune enhancer, which comprises one or more polypeptides having the structure of Formula I.
  • the tumor immune enhancer of the present invention can enhance the specific CD8 + CTL immune response of the tumor neoantigen vaccine, and has the activity of enhancing the anti-tumor immune response of the tumor vaccine.
  • the tumor immune enhancer of the present invention includes the polypeptide shown in SEQ ID NO.: 1-3 or a polypeptide derived therefrom.
  • Neoantigen The protein with specific amino acid sequence variation produced by cancer cells on the basis of gene variation is called "neoantigen" (NeoAg). This is because if there is no change in the amino acid sequence, these proteins should not be antigenic. Once mutated, these proteins will attract the attention of autoimmune cells and cause a series of immune responses.
  • the tumor immune enhancer of the present invention can enhance the immune response of tumor neoantigens, especially antigens that cannot effectively trigger anti-tumor immune responses.
  • the tumor antigen can be natural, artificially synthesized, or a combination thereof.
  • the tumor antigen can be a short peptide, an intact protein, a tumor cell lysate, or a combination thereof.
  • melanoma cells B16F10 neoantigen peptides (NeoAg) are used as vaccines, which cannot produce antigen-specific anti-tumor CTLs when administered alone.
  • the cell lysate involved in the present invention is to suspend the cells in an equal volume of phosphate buffer, freeze-thaw the cells to rupture, and centrifuge at 10,000 g for 10 minutes to remove the precipitate and use the supernatant as the cell lysate.
  • the present invention also relates to polynucleotides encoding DTHE polypeptides.
  • a preferred coding sequence encodes the short peptide shown in SEQ ID NO: 1-9.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA can be a coding strand or a non-coding strand.
  • the full-length sequence of the polynucleotide of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • the DNA sequence encoding the polypeptide (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector or UTHE polypeptide coding sequence of the present invention.
  • the polypeptide of the present invention can be a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptide of the present invention can be chemically synthesized or recombinant.
  • the polypeptide of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.
  • the UThE polypeptide with 6 Arg at the end can be synthesized by chemical synthesis, the HPLC purification yield is greater than 20%, and the purity is greater than 99.9%.
  • a preferred method is to use liquid phase synthesis technology or solid phase synthesis technology, such as Boc solid phase method, Fmoc solid phase method or a combination of the two methods.
  • Solid-phase synthesis can quickly obtain samples, and an appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide.
  • the preferred solid-phase carrier in the Fmoc system is the Wang resin connected with the C-terminal amino acid in the peptide.
  • the Wang resin structure is polystyrene, and the arm between the amino acid is 4-alkoxybenzyl alcohol; 25% hexahydropyridine is used /Dimethylformamide is treated at room temperature for 20 minutes to remove the Fmoc protecting group and extend from the C-terminus to the N-terminus one by one according to the given amino acid sequence.
  • the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol and the protective group is removed. After the resin is filtered off, the crude peptide can be separated by ether precipitation and separation.
  • the desired peptide is purified by gel filtration and reverse phase high pressure liquid chromatography.
  • the preferred resin is a PAM resin connected with the C-terminal amino acid in the peptide.
  • the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxymethyl benzene acetamide; synthesized in Boc
  • the protective group Boc is removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane.
  • the peptide chain condensation is completed Afterwards, use hydrogen fluoride (HF) containing p-cresol (5-10%) at 0°C for 1 hour to cut the peptide chain from the resin while removing the protective group.
  • HF hydrogen fluoride
  • acetic acid containing A small amount of mercaptoethanol
  • the solution is lyophilized and further separated and purified with molecular sieve Sephadex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide.
  • Various couplings known in the field of peptide chemistry can be used Reagents and coupling methods to couple each amino acid residue, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetraurea hexafluorophosphate can be used (HBTU) for direct coupling.
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • HBTU 1,1,3,3-tetraurea hexafluorophosphate
  • Another method is to use recombinant technology to produce the polypeptide of the present invention.
  • the polynucleotides of the present invention can be used to express or produce recombinant DTHE polypeptides.
  • the recombinant polypeptide can be expressed in the cell, on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • polypeptide of the present invention is relatively short, it can be considered to connect multiple polypeptides in series, obtain the expression product in the form of multimers after recombinant expression, and then form the required small peptides by methods such as restriction enzyme digestion.
  • the present invention also provides a pharmaceutical composition, which can be therapeutic or prophylactic (such as a vaccine).
  • the pharmaceutical composition of the present invention contains (a) a safe and effective amount of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient.
  • an effective dose is about 10 micrograms-100 mg/dose, preferably 100-1000 micrograms/dose of the polypeptide of the present invention administered to an individual.
  • the polypeptide of the present invention can be used alone or together with other therapeutic agents (for example, formulated in the same pharmaceutical composition).
  • the preventive pharmaceutical composition may be a vaccine composition, which comprises the polypeptide of the present invention and a tumor antigen, and is usually combined with a "pharmaceutically acceptable carrier".
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent.
  • the term refers to medicament carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and do not have excessive toxicity after administration.
  • Such vectors are well known to those of ordinary skill in the art.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants and combinations thereof.
  • these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
  • the (vaccine) composition of the present invention may also contain additional adjuvants.
  • vaccine adjuvants include (but are not limited to) the following types: inorganic adjuvants, such as aluminum hydroxide, alum, etc.; synthetic adjuvants, such as synthetic double-stranded polynucleotides (double-stranded polyadenosine Acid, uridine acid), levamisole, isoprinosine, etc.; oil agents, such as Freund’s adjuvant, peanut oil emulsification adjuvant, mineral oil, vegetable oil, etc.;
  • Adjuvants also include various new adjuvants and adjuvant components that are considered to be useful as vaccine adjuvants in current and future studies.
  • the vaccine composition or immunogenic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension or a liquid excipient before injection.
  • the preparation can also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.
  • composition can be made into a unit or multiple dosage form.
  • Each dosage form contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, and appropriate pharmaceutical excipients.
  • composition of the invention can be administered by conventional routes, including (but not limited to): intravenous, intratumor, intramuscular, intraperitoneal, subcutaneous, intradermal, para-cancerous, or topical administration. medicine.
  • the object to be prevented or treated can be an animal; especially a human.
  • compositions of different dosage forms can be used according to the use situation.
  • These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained-release formulation.
  • the short peptide DTHE or its salt can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the dosage of the short peptide DTHE or its pharmaceutically acceptable salt as the active ingredient can be adjusted according to the weight, age, sex, and degree of symptoms of each patient to be treated.
  • the present invention utilizes composite antigen carrier technology to recognize tumor neo-antigens to the greatest extent;
  • polypeptide of the present invention can improve the efficiency of immune cell activation in vitro.
  • neoAg neoantigenic peptide
  • amino acid sequences of the four neoantigen peptides are as follows:
  • the cells were suspended in an equal volume of phosphate buffer. After freezing and thawing to rupture the cells, the supernatant was used as cell lysate after centrifugation at 10,000 g for 10 minutes to remove the precipitate.
  • As tumor antigens B16F10 cell lysates, LLC cell lysates, and Hepa1-6 cell lysates were prepared respectively.
  • Example 1 UThE enhances the anti-tumor immune response of melanoma weak neoantigen
  • neoantigen peptides from the B16F10 cell line that have been shown to be ineffective in inducing anti-tumor immune responses in mice were selected by preclinical studies.
  • B16F10 is a mouse melanoma cell.
  • neoAg peptides #1, #2, #3, and #4 were mixed at a weight ratio of 1:1:1:1 and formulated into a vaccine together with UThE peptide and adjuvant. After immunizing mice, their anti-tumor effects were tested .
  • the specific method is as follows:
  • C57BL6 mice (6 weeks old, 7 in each experimental group) were immunized three times, one week apart. Each mouse used 200 microliters of vaccine for each immunization, and 50 microliters were injected subcutaneously near the side of the limbs.
  • the dosage of each component of 200 microliters vaccine is neoAg50 micrograms, DThE50 micrograms, Alum300 micrograms, and CpG20 micrograms.
  • the vaccine injection is prepared with PBS.
  • mice On the 7th day after inoculation, the mice began to develop tumors. On the 15th day, the average tumor volume (mean ⁇ SEM) of mice in each group of PBS, NeoAg, NeoAg+UThE was 785 ⁇ 153mm 3 , 890 ⁇ 80mm 3 , and 328 ⁇ 65mm 3 respectively .
  • Example 2 UThE enhances the anti-tumor immune response of melanoma weak neoantigen
  • the method of this example is basically the same as that of example 1, but the difference lies in the preparation of the vaccine and the number of immunizations, using B16F10 cell lysate or neoAg as the tumor antigen. Methods as below:
  • C57BL/6 mice (6 weeks old, 5 in each experimental group) were immunized four times with one week interval each time. Each mouse was injected with the vaccine at four sideways positions close to the extremities, each injection of 50 microliters.
  • the dosage of vaccine components for the first and second vaccines per 200 microliters is 25 micrograms of neoAg or B16F10 cell lysate, 25 micrograms of UThE 25 micrograms adjuvant (adj), and 100 microliters of MF59100 microliters.
  • Adjuvants include 12.5ugCpG, 12.5 ⁇ g PolyI:C, formulated with PBS.
  • the dosage of each component of the vaccine per 200 microliters is 12.5 micrograms of neoAg or B16F10 cell lysate, 12.5 micrograms of UThE, 12.5 micrograms of adjuvant (adj), and 59 microliters of MF.
  • Adjuvants include 6.25 micrograms of CpG and 6.25 micrograms of PolyI:C, formulated with PBS. The application status of each group is as follows:
  • mice were inoculated subcutaneously near the right armpit 105 B16F10 cells. The cells were suspended in 100 microliters of PBS. Then observe and record the tumor growth. After the tumor is visible, the tumor volume is measured every other day.
  • mice in the Lys+UThE+Adj group had no tumors on the 19th day.
  • mice in the Neo+UThE+Adj group also had smaller tumors and slower growth (Figure 2A).
  • the mean tumor volume (mean ⁇ SEM) of the adj group, UTEh+adj group, neo+adj group, neo+UThE+adj group and Lys+UThE+Adj group were 801.1 ⁇ 821.7mm 3 , respectively. 517.4 ⁇ 615.5mm 3 , 431.8 ⁇ 886.7mm 3 , 317.8 ⁇ 314.4mm 3 ( Figure 2B).
  • Example 3 effectively enhances the therapeutic effect on melanoma B16F10
  • Examples 1-3 it has been demonstrated that pre-administration of UThE peptide can be used as an immune enhancer to enhance the anti-tumor effect of tumor antigen peptides.
  • UThE peptide can be used as an immune enhancer to enhance the anti-tumor effect of tumor antigen peptides.
  • the method is as follows: simulate tumor treatment, first inoculate tumor cells, and then treat with UThE immunopotentiator.
  • B16F10 cells 1*10 5 were inoculated into the right forelimb of the mouse near the armpit, and 5 days later, the mice were immunized. Each mouse was vaccinated with 100 microliters, divided into two sideways close to the hindlimb injection site, each point was 50 microliters. The dosage of each component of the vaccine per 200 microliters is: 25 micrograms of UThE, 100 microliters of adjuvant Adj, and MF59. Adjuvants include CpG12.5 micrograms, PolyI:C25 micrograms, formulated with PBS. A total of four immunizations, one week apart each time. After the tumor began to appear, the size of the tumor was measured and the weight of the mouse was weighed.
  • Example 4 enhances the detection of humoral immune response of neoantigen peptide NeoAg by ELISA
  • the immunized serum of the mice was diluted with 0.1% BSA in PBS at a volume ratio of 1:100. Add 100 microliters of neoantigen peptide solution (10 micrograms/ml pH9.5 carbonic acid buffer) to the 96-well plate, and coat overnight at 4°C. Then, the 96-well plate was blocked with 0.1% BSA in PBS for 2 hours at room temperature.
  • Figure 4 shows the results of ELISA detection of UThE antibody in serum samples of immunized mice.
  • Mice were immunized with DThE+CpG+polyI:C+MF59 vaccine four times, with an interval of one week each time, and vaccinated with 7.5*10 4 B16F10 cells 10 days after immunization.
  • the tumor volume of mice reached 1500mm 3 or on the 30th day after tumor inoculation.
  • the mice were sacrificed and blood was collected. Because IgG1 and IgG2 are present at the same time and their concentration ratio is greater than 1, the T helper cell response is a mixed type I + type II, and the type II response is stronger.
  • Figure 5 shows the results of ELISA detection of UThE antibodies in serum samples of immunized mice.
  • Mice were immunized four times with neoAg+UThE+CpG+polyI:C+MF59 vaccine, with an interval of one week each, and vaccinated with 7.5*10 4 B16F10 cells 10 days after immunization.
  • the tumor volume of mice reached 1500mm 3 or the 30th after tumor inoculation. Day, the mice were sacrificed and blood was collected. Because IgG1 and IgG2 exist at the same time and their concentration ratio is greater than 1, the T helper cell response is type I + type II mixed, and type II response is stronger.
  • Figure 6 shows the results of ELISA detection of neoAg antibodies in serum samples of immunized mice.
  • Mice were immunized with neoAg+CpG+polyI:C+MF59 vaccine four times, with a one-week interval each time, and vaccinated with 7.5*10 4 B16F10 cells 10 days after immunization.
  • the tumor volume of mice reached 1500mm 3 or on the 30th day after tumor inoculation.
  • the mice were sacrificed and blood was collected.
  • the antibodies produced are mainly IgG2, therefore, the T helper cell response is type I.
  • Figure 7 shows the results of ELISA detection of neoAg antibodies in serum samples of immunized mice.
  • Mice were immunized four times with neoAg+UThE+CpG+polyI:C+MF59 vaccine, with an interval of one week each, and vaccinated with 7.5*10 4 B16F10 cells 10 days after immunization.
  • the tumor volume of mice reached 1500mm 3 or the 30th after tumor inoculation. Day, the mice were sacrificed and blood was collected. Because IgG1 and IgG2 exist at the same time and their concentration ratio is greater than 1, the T helper cell response is type I + type II mixed, and type II response is stronger.
  • Example 5 UThE polypeptide promotes neo-Ag specific CD8+CTL cell response
  • the method is as follows: After immunized mouse spleen lymphocytes are incubated with UThE or neoAg antigen presenting cells for 5 days, the lymphocytes are purified using T lymphocytes or CD8+T separation kit. In the INF- ⁇ coated with the capture antibody immunoblot was added 100 microliters of a 96 well plate containing 10 4 lymphocytes purified broth. Incubate at 37°C and 5% CO 2 for 16 hours. Then the immunospot kit was used to detect the number of T cells secreting INF- ⁇ . After the T effector cells were co-cultured with tumor target cells at 20:1, LDH was used to detect cell killing.
  • UThE or neoAg antigen presenting cell dendritic cell preparation method Take healthy mice of the same strain after euthanasia, separate the cortex, take the front and rear leg bones, cut off both ends of the leg bones with scissors, and use PBS (containing 1%) FBS) 1ml syringe, aim the needle at the red spot on one end of the leg bone, and flush out the bone marrow cells inside with PBS. Use a pipette to gently blow away the tissue pieces, pass through a 200-mesh sieve and transfer to a 50ml centrifuge tube, and centrifuge at 400g for 10 minutes. Resuspend the cells in PBS, add red blood cell lysate, and centrifuge at 400g for 15 minutes.
  • PBS containing 1%) FBS 1ml syringe
  • Preparation method of mouse spleen lymphocytes Take the mouse spleen of the experimental group, grind and smash the culture medium and wash it through a 200-mesh screen, 400g, 8min, centrifuge to remove the supernatant, remove the red blood cells from the red blood cell lysate, resuspend the cells in PBS, count, and culture Add IL2 (20ng/ml) to the base to continue the culture.
  • the method of this example is basically the same as Example 1 and Example 2.
  • the difference lies in the addition of lung cancer cells LLC, prostate cancer cells RM-1, and pancreatic cancer cells PanC-02 based on the experimental basis of the protomelanoma cell B16F10.
  • the preparation and the number of immunizations are basically the same, and the tumor antigen of each cell lysate is used. Methods as below:
  • C57BL/6 mice (6 weeks old, 4-6 mice in each experimental group) were immunized four times with one week interval each time. Each mouse was injected subcutaneously at 2 sites on the roots of the hind limbs, with 50 microliters injected per point. The doses of the four immunizations were the same. The amount of vaccine components per 50 microliters was 25 micrograms of cell lysate and 25 micrograms of UThE. The adjuvants included 25 microliters of MF59, 25ugCpG, and 12.5 micrograms of PolyI:C, prepared with PBS.
  • mice On the third day after the fourth immunization, the mice were inoculated subcutaneously near the right side of the armpit.
  • LLC cell inoculation volume was 1*10 5
  • RM-1 cell inoculation volume was 1 *10 5 .
  • the cells were suspended in 100 microliters of PBS. Then observe and record the tumor growth. After the tumor is visible, the tumor volume is measured every other day.
  • the results of lung cancer LLC cell inoculation are shown in Figure 11 and Table 4.
  • the order of tumor growth rate and volume size was: Lys+adj group>Lys+Th+Adj group.
  • the tumor size on day 17 is shown in Figure 11.
  • the average tumor weight (mean ⁇ SEM) is 0.4475 ⁇ 0.04211g and 0.235 ⁇ 0.02958g, respectively.
  • melanoma B16F10 cell inoculation The results of melanoma B16F10 cell inoculation are shown in Figure 12 and Table 5. From the injection of tumor cells to the 24th day, the order of tumor growth rate and volume size was: Lys+adj group>Lys+Th+Adj group. Day 17 in tumor size 12, the mean tumor volume (mean ⁇ SEM) were: 419.9 ⁇ 165mm 3, 154.9 ⁇ 111.6mm 3.
  • the results of inoculation of prostate cancer RM-1 cells are shown in Figure 13 and Table 6.
  • the order of tumor growth rate and volume size was: Adj group>Th+Adj group>Lys+Adj group>Lys+Th+Adj group.
  • the tumor size on day 24 is shown in Figure 13.
  • the mean tumor volume (mean ⁇ SEM) is 1476 ⁇ 436.9mm 3 , 1457 ⁇ 377.1mm 3 , 1335 ⁇ 384.6mm 3 , and 881.3 ⁇ 301.3mm 3 respectively .
  • PanC-02 cells of pancreatic cancer are shown in Figure 14 and Table 7.
  • the order of tumor volume size was: Lys+adj group>Th+adj group>Lys+Th+Adj group.
  • the mean tumor volume (mean ⁇ SEM) was 0.4475 ⁇ 0.04211mm 3 and 0.235 ⁇ 0.02958mm 3 respectively .
  • the method of this embodiment is basically the same as that of Embodiment 1, Embodiment 2, and Embodiment 6.
  • the experimental protocol is a B16F10 tumor treatment model, and only low to high doses of UThE combined with the adjuvant Adj are used to treat tumors in mice.
  • Each mouse was injected subcutaneously at 2 sites on the roots of the hind limbs, with 50 microliters injected per point.
  • the doses of the four immunizations are the same, and the dosage of each 50 microliter vaccine component is adjuvant (including 25 microliters MF59, 25ugCpG, 12.5 microgram PolyI:C) and UThE, and the doses of UThE are 30 micrograms, 50ug and 80ug, respectively.
  • the results of tumor emergence in each treated group are shown in Figure 15 and Table 8.
  • the order of tumor growth rate and volume size was: PBS group>Adj group>Adj+Th-30ug group>Adj+Th -50ug group>Adj+Th-80ug group.
  • the mean tumor volume (mean ⁇ SEM) on day 18 was 480.4 ⁇ 118.6mm 3 , 309.9 ⁇ 139.6mm 3 , 253.3 ⁇ 70.59mm 3 , 115.8 ⁇ 45.05mm 3 , 5.753 ⁇ 5.743mm 3, respectively .
  • Example 8 The application method is the same as that of Example 7, but different from that of Example 7 to investigate the dose-effect relationship of UThE.
  • the purpose of Example 8 is to investigate the inhibitory effect of each polypeptide on tumors of different strains of mice at the same dose of 50 ug.
  • the dosage of each peptide of DThE1-7 is 50ug, and the dosage of adjuvant is the same as in Example 7.
  • C57BL/6 mice were inoculated with 10 5 B16F10 cells, Balb/C mice were inoculated with 10 4 4T1 cells, and immunized on the third day after tumor inoculation.
  • the tumor growth of mice in each group is shown in Figure 16.
  • mice have different anti-tumor effects on the polypeptides, which may be related to the MHC II type of the two strains of mice, suggesting that MHC-related typing should be considered in the later administration, and targeted immunization can be carried out.

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Abstract

L'invention concerne un activateur immunitaire tumoral, une composition pharmaceutique de celui-ci et un procédé de préparation associé. L'activateur comprend un ou plusieurs polypeptides utilisant des séquences représentées dans SEQ ID NO. : 1 à 9 en tant que structures de noyau, et peut être utilisé pour préparer des vaccins antitumoraux.
PCT/CN2021/078264 2020-02-28 2021-02-26 Activateur immunitaire tumoral, son procédé de préparation et son utilisation WO2021170111A1 (fr)

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JP7513303B2 (ja) 2024-07-09
JP2023515850A (ja) 2023-04-14
CN113318225B (zh) 2024-01-19
US20230045104A1 (en) 2023-02-09
EP4112636A4 (fr) 2023-09-27
CA3169907A1 (fr) 2021-09-02

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